Basal Medium Research Articles

Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.

A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.

IN-VITRO REGENERATION OF MULTIPLE SHOOTS FROM APICAL MERISTEMS OF COWPEA (Vigna unguiculata L. (WALP) USING DIFFERENT GENOTYPES (LOCAL AND IMPROVED) AND MEDIA COMPOSITIONS

Organogenesis in-vitro was evaluated in explants derived from shoot apices of four cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate multiple shoots, apices of the in vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine(BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. The highest shoot number was recorded with IT99k-573-2-1 and Danilla and shoot length was highest in Kanannado, IT04k-33-2-1and Danilla indicating that Danilla responded well to in vitro regeneration. To optimise the production of multiple shoots the effects of different concentratios (0.5, 1.0 and 1.5mg/L) of 6-benzyaminopurine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid on the number of responding explant, number of multiple shoot produced and length of the shoots were tested. after six weeks of culture, the percentage of responding shoot apices was highest on media supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.The number of responding apices decreasing with decrease in the concentration of BAP. Maximum number of multiple shoots and the highest mean shoot length were also obtained when MS was supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.

One-step in vitro protocol for clonal propagation of Dendrobium Yuki White, a high value ornamental orchid hybrid

A novel and accelerated one-step protocol for complete in vitro mass propagation of Dendrobium Yuki White was optimized for the first time. Rapid shoot-root initiation and multiplication was simultaneously achieved from apical shoot segment explants on Murashige and Skoog basal medium fortified with 0.5 mg L−1 N6-benzyladenine (BA), 0.1 mg L−1 α-naphthaleneacetic acid, along with 40 mg L−1 adenine sulphate as an additive. Maximum number of shoots per explant (12) was obtained within eight weeks of culture. Maximum number of root induction (17) and elongation (6.5 cm) were recorded within this period. On the other hand, the matrix plot, network plot, and principal component analyses illustrated the association of auxin-cytokinin and the multiple growth attributes under study. In a simple acclimatization procedure, involving primary acclimatization in charcoal blocks for six weeks, and followed by a combination of coconut fibre and charcoal (1:1; v v−1) with intermittent water spraying ensured 97% survival rate in the next ten weeks. Based on the comprehensive information developed in this study, this protocol should be of immense importance for large-scale production of planting materials as well as for exploring the possible comparative roles of the auxin-cytokinin combinations used in this study.

SOMATIC EMBRYOGENESIS IN LOCAL COWPEA (Vigna unguiculata L. (WALP) VARIETIES

Organogenesis in vitro was evaluated in explants derived from shoot apices of two local cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate shoots, apices of the In Vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. Variation was observed in response of the shoot apices from different cowpea cultivars to in vitro regeneration of shoots. The highest shoot number was recorded with Danilla and shoot length was highest in Kanannado, indicating that Danilla responded well to in vitro regeneration. While there were significant differences between these concentrations and other concentrations used in this study on the number of shoots produced, statistically there was no difference between the traetments tested on the in vitro shoot length recorded in this study.Although shoots were succesfully regenerated in this study rooting was not achieved as the explants gradually transformed into calli

Insulin enhances neurite extension and myelination of diabetic neuropathy neurons.

BackgroundThe authors established an in vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons.MethodsSCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination.ResultsThe cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media.ConclusionsInsulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy.

In vitro evaluation of porous poly(hydroxybutyrate-co-hydroxyvalerate)/akermanite composite scaffolds manufactured using selective laser sintering.

Incorporation of a bioactive mineral filler in a biodegradable polyester scaffold is a promising strategy for scaffold assisted bone tissue engineering (TE). The current study evaluates the in vitro behavior of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV)/Akermanite (AKM) composite scaffolds manufactured using selective laser sintering (SLS). Exposure of the mineral filler on the surface of the scaffold skeleton was evident from in vitro mineralization in PBS. PHBV scaffolds and solvent cast films served as control samples and all materials showed preferential adsorption of fibronectin compared to serum albumin as well as non-cytotoxic response in human osteoblasts (hOB) at 24h. hOB culture for up to 21days revealed that the metabolic activity in PHBV films and scaffolds was significantly higher than that of PHBV/AKM scaffolds within the first two weeks of incubation. Afterwards, the metabolic activity in PHBV/AKM scaffolds exceeded that of the control samples. Confocal imaging showed cell penetration into the porous scaffolds. Significantly higher ALP activity was observed in PHBV/AKM scaffolds at all time points in both basal and osteogenic media. Mineralization during cell culture was observed on all samples with PHBV/AKM scaffolds exhibiting distinctly different mineral morphology. This study has demonstrated that the bioactivity of PHBV SLS scaffolds can be enhanced by incorporating AKM, making this an attractive candidate for bone TE application.

An efficient protocol optimization for callus induction, regeneration and acclimatization in sugarcane cv. CO 94012

Sugarcane's vast and complex genome, photosensitivity, low seed setting, prolonged crop duration, and complex environmental effect make it difficult to create diverse genetic variability by conventional breeding. However, the in vitro culture system can play a vital role in boosting yield, quality, and making the crop climate smart through in vitro approaches. In the present study in vitro culture using immature leaf sheath of Sugarcane (Saccharum officinarum L. cv- CO 94012) as an ex plant was used to standardize the protocol for callus induction and whole plant regeneration. MS medium supplemented with 2.0 mg/l, 2-4 D, recorded the highest percentage of callus induction. The highest number of multiple shoots was produced on MS medium fortified with KIN 1.0 mg/l. Rooting was more profuse when in vitro regenerated shoots were inoculated on MS basal medium supplemented with 3.0 mg/l NAA. Rooted shoots were hardened in the greenhouse before being transplanted to the field, where 90% survival rate.

Polystyrene shaping effect on the enriched bacterial community from the plastic-eating Alphitobius diaperinus (Insecta: Coleoptera)

Plastic pollution has become a serious issue of global concern, and biodegradation of plastic wastes is representing one attractive environment-friendly alternative to traditional disposal paths. It is known that insects are involved in the plastic polymer degradation process, with reported evidence of tenebrionid beetle larvae capable to degrade polystyrene (PS), one of the most used plastics worldwide. Recently, a ribosomal RNA based survey on the insect gut microbiota of the lesser mealworm Alphitobius diaperinus has revealed differentially abundant microbial taxa between PS-fed larvae and control group. Following these findings, an enrichment bacterial culture was set up in liquid carbon-free basal medium with PS film as sole carbon source using PS-fed larvae of A. diaperinus as inoculum. After two-months the culture was analysed both by molecular and culture-based methods. Isolated bacteria which had become prevalent under the selective enrichment conditions resulted ascribable to three taxonomic groups: Klebsiella, Pseudomonas, and Stenothrophomonas. The predominance of these groups in PS-fed larvae was confirmed by using bacterial 16S rRNA gene amplicon sequencing, and it was consistent with the results of previous reports. Isolated bacteria were able to attach to PS surfaces and SEM observations showed the presence of thin fibrillar structures connecting the bacterial cells to the abiotic surface.

Airway Fibroblast Secretory Products Enhance Cell Migration

Background: The nasal fibroblast secretome, which includes various cytokines, chemokines, and growth factors, promotes cell migration. Currently, the proteomics of Airway Fibroblast (AF) Conditioned Medium (AFCM) are being actively studied. Objective: This study was aimed at profiling and identifying the AF secreted proteins that can enhance wound healing of the airway epithelium and predict the potential pathway involved. Methods: Airway Epithelial Cells (AECs) and AFs were isolated from redundant human nasal turbinate and cultured. AFCM was collected by culturing the AFs either with serum-free airway epithelium basal medium (AECM) or with serum-free F12:DMEM (FDCM). For evaluating cell migration, the AECs were supplemented with airway epithelium medium and defined keratinocyte medium (1:1; AEDK; control), or with AEDK supplemented with 20% AECM or 20% FDCM. The mass spectrometry sample was prepared by protein precipitation, followed by gel electrophoresis and in-gel digestion. Results : AECM promoted better cell migration compared to the FDCM and the control medium. Bioinformatics analysis identified a total of 121, and 92 proteins from AECM and FDCM, respectively: 109 and 82 were identified as secreted proteins, respectively. STRING® analysis predicted that 23 proteins from the AECM and 16 proteins from the FDCM are involved in wound healing. Conclusion: Conditioned medium promotes wound healing by enhancing cell migration, and we successfully identified various secretory proteins in a conditioned medium that play important roles in wound healing.

Genetic regulation and fermentation strategy for squalene production in Schizochytrium sp.

Squalene, as an important terpenoid, is extensively used in the medicine and health care fields owing to its functions of anti-oxidation, blood lipid regulation and cancer prevention. The marine microalgae, Schizochytrium sp., which acts as an excellent strain with potential of high squalene production was selected as the starting strain. The overexpressed strain with sqs gene got the reduced biomass and lipid, while the squalene titer was increased by 79.6% ± 4.7% to 12.8 ± 0.2mg/L. In order to further increase squalene production, the recombinant strain (HS strain) with sqs and hmgr gene co-overexpression was further constructed. The biomass and squalene titer of the HS strain were increased by 13.6% ± 1.2% and 88.8% ± 5.3%, respectively, which indicated the carbon flux of the mevalonate pathway was enhanced for squalene accumulation. Regarding the squalene synthesis is completely coupled with cell growth, fermentation strategy to prolong the logarithmic growth phase was conducive to improve squalene production. Under the condition of optimal composition and concentrated medium, the squalene titer of HS strain was 27.0 ± 1.3mg/L, which was 2.0 times that of the basal medium condition (13.5 ± 0.4mg/L). This study which combined the metabolic engineering and fermentation strategy provides a new strategy for squalene production in Schizochytrium sp. KEY POINTS: •The overexpression of sqs and hmgr genes promoted carbon metabolism for squalene. •The optimal and concentrated media can increase squalene yield.

Multiple Shoot Regeneration from Detached Embryonic Axis in Greengram (Vigna radiata) cv.SML 668

Background: Under prevailing regime of climate change and dynamic pest-crop interactions, greengram genetic improvement requires a speedier and precise tool such as genetic transformation to develop improved lines. In this context, an in vitro multiple shoot regeneration system via organogenesis was demonstrated in mungbean cv. SML668, a parent cultivar used in crop improvement programs, using double cotyledonary node (DCN) and detached embryonic axis (EA) explants with 1.0 mg l-1 BAP. Methods: While both the explants responded to in vitro regeneration, number of shoots regenerated was higher with EA (4.02±0.19) than with DCN (3.1±0.08). 6-benzyl aminopurine (BAP) was found to be most effective for inducing and regenerating multiple shoots, in comparison to all other phytohormones (NAA, IAA and TDZ) and supplements (amino acids) tested. Sub culturing twice on BAP supplemented media followed by two subcultures on basal media was optimal for multiple shoot regeneration. Rhizogenesis was obtained on basal media devoid of any phytohormones in EA explants and in 1.0 mg l-1 IAA for DCN explants. The in vitro regenerated plantlets were successfully hardened in a mixture of soil, sand and vermiculite that flowered, produced pods and viable seeds on maturity. Result: The study revealed that detached embryonic axis was a potential explant for in vitro regeneration in SML668, a cultivar not tested for its in vitro regeneration ability before.

Comparison of Liquid and Solid-State Fermentation Processes for the Production of Enzymes and Beta-Glucan from Hulled Barley.

Solid-state fermentation using hulled barley was carried out to produce enzymes and β-glucan. The one-factor-at-a-time experiments were carried out to determine the optimal composition of the basal medium. The modified synthetic medium composition in liquid-state fermentation was determined to be 70 g/l hulled barley, 0 g/l rice bran, 5 g/l soytone, and 6 g/l ascorbic acid. Optimal pretreatment conditions of hulled barley by solid-state fermentation were evaluated in terms of maximum production of fungal biomass, amylase, protease, and β-glucan, which were 1.26 mg/g, 31310.34 U/g, 2614.95 U/g, and 14.6% (w/w), respectively, at 60 min of pretreatment condition. Thus, the solid-state fermentation process was found to enhance the overall fermentation yields of hulled barley to produce high amounts of enzymes and β-glucan.

Biotechnological use of dairy by-products for the production and microencapsulation of the food preservative enterocin CRL35.

Bacteriocins from Gram-positive bacteria have been proposed as natural food preservative and there is a need for large-scale production for commercial purposes. The aim of the present work is to evaluate whey, a cheese industrial by-product, for the production and microencapsulation of enterocin CRL35. Whey proved to be a promising basal medium for bacterial growth although the bacteriocin production was quite low. However, it could be much favored with the addition of yeast extract at concentrations as low as 0.5%. Besides improving bacteriocin production, this peptide was successfully microencapsulated by spray drying using whey protein concentrate and a chitosan derivative as wall materials. Microcapsules averaging 10± 5μm diameter were obtained, with good structural integrity and high antimicrobial activity with a stability of at least 12 weeks at 4°C.Insummary, sustainable bacteriocin production and microencapsulation was achieved recycling whey or its derivatives. In addition, the formulation owns high antimicrobial activity with a long shelf life. The development of a food preservative may represent a green solution for handling whey.

Combined inorganic nitrogen sources influence the release of extracellular compounds that drive mutualistic interactions in microalgal‒bacterial co-cultures

We investigated the role of extracellular metabolites released during mutualistic interactions in co-cultures of a microalga, Tetradesmus obliquus IS2 or Coelastrella sp. IS3, and a bacterium, Variovorax paradoxus IS1, grown with varying levels of NO3–N and NH4–N. Both NO3–N and NH4–N were added to modified Bold’s basal medium at 16:0, 12:4, 8:8; 4:12 and 0:16 molar ratios by keeping a final N:P ratio of 16:1. Monocultures of microalgae grown with nitrate alone showed enhanced growth (> twofold) than ammonium, while the bacterial strain cultured with ammonium alone exhibited a > 1.3-fold increase in growth than nitrate. Co-culturing performed higher growth at combined nitrate and ammonium supply as compared to the single cultures. The same ratio of nitrate and ammonium resulted in superior growth of microalgae (> 1.7-fold) and the bacterium (> 4.1-fold) as compared to the monocultures. Uptake of NO3–N, NH4–N and PO4–P by monocultures or co-cultures depended on the ratio of two inorganic nitrogen sources used. The composition of organic acids, amino acids and simple sugars in exudates from monocultures varied with the ratios of nitrate and ammonium in the medium. Thus, the present novel study demonstrates that the release of exudates is affected both qualitatively and quantitatively during mutualistic interactions in microalgal‒bacterial co-cultures under the impact of inorganic nitrogen sources. Our results suggest that the variables such as inorganic nitrogen sources and extracellular metabolites released need to be considered while using co-cultures for effective bioremediation of wastewaters.

Biological Activity and NMR-Fingerprinting of Balkan Endemic Species Stachys thracica Davidov.

Stachys thracica Davidov is a Balkan endemic species distributed in Bulgaria, Greece, and Turkey. In Bulgaria, it is classified as “rare” and is under the protection of the Bulgarian biodiversity law. The aim of our study was to develop an efficient protocol for ex situ conservation of S. thracica and to perform comparative NMR-based metabolite profiling and bioactivity assays of extracts from in situ grown, in vitro cultivated, and ex vitro acclimated plants. Micropropagation of S. thracica was achieved by in vitro cultivation of mono-nodal segments on basal MS medium. Ex vitro adaptation was accomplished in the experimental field with 83% survival while conserved genetic identity between in vitro and ex vitro plants as shown by the overall sequence-related amplified polymorphism marker patterns was established. Verbascoside, chlorogenic acid, and trigonelline appeared the main secondary metabolites in in situ, in vitro cultivated, and ex vitro acclimated S. thracica. High total phenolic and flavonoid content as well as antioxidant and radical scavenging activity were observed in in situ and ex vitro plants. Further, the anti-inflammatory activity of S. thracica was tested by hemolytic assay and a high inhibition of the complement system was observed. Initiated in vitro and ex vitro cultures offer an effective tool for the management and better exploitation of the Stachys secondary metabolism and the selection of lines with high content of bioactive molecules and nutraceuticals.

Evaluation of Sugar Mill By-product Molasses as a Low Cost Culture Media for Microalgae

Microalga Chlorella vulgaris was cultured in different concentrations of normal molasses medium (NMM0.5 g/l, NMM1.0 g/l, and NMM1.5 g/l) to evaluate growth performance of the sugar mill byproduct as a low cost culture media and Bold basal medium (BBM) as control. Maximum growth of Chlorella vulgaris was found in NMM 1.0 g/l on 8th day of the culture followed by BBM, NMM 0.5 g/l and NMM 1.5 g/l. Similar findings were also observed in determining chlorophyll a content and optical density of C. vulgaris. Maximum cell growths 191.88 (Χ 105)/mL, chlorophyll a content 10.60 (mg/l) and optical density 2.15 were recorded in NMM1.0 g/l. Maximum SGR of cell was determined 0.56 (mg/day) grown in NMM1.0 g/l followed by 0.52, 0.52 and 0.48 (mg/day) in BBM, NMM0.5 g/l and NMM1.5 g/l, respectively. Chlorophyll a content and total biomass of Chlorella vulgaris followed the similar trend. Protein (46.49%) and lipid (14.18%) of C. vulgaris was detected significantly higher (P<0.01) in NMM1.0 g/l than that grown in BBM and other concentrations of NMM. The growth performance of the investigating molasses medium (NMM1.0 g/l) indicates that the molasses may be a good low cost culture medium ingredient source for C. vulgaris or any other microalga species.

Keratinolytic bacteria from the feathers of wild Dark-eyed Juncos (Junco hyemalis)

We collected >300 bacterial isolates from overwintering Dark-eyed Juncos ( Junco hyemalis) to find that ∼40% appear to degrade the structural protein of feathers using extracellular keratinase enzymes. A guild of bacteria (∼18% of total counts) grew significantly better on basal salt medium (BSM) plates containing feather meal than on BSM agar-only plates (∼8% total counts). The genus-level profile of the 107 most active keratinolytic bacterial isolates shows that nearly half are Bacillus species. Keratinolytic Frigoribacterium, Microbacterium, Okibacterium, Pantoea, Sphingomonas, Staphylococcus, and Stenotrophomonas were found, among others. Active isolates were shown to degrade whole feathers, used feather meal as a sole carbon and energy source, and produced zones of clearing indicative of extracellular proteases on milk agar plates. Our novel in vitro keratinase assay uses ninhydrin to quantitate the release of amino acids from exogenously added keratin, with Bacillus licheniformis str. ATCC 14580 serving as a positive control. Keratinolytic isolates were grown in the presence of feather meal to induce keratinase release and this conditioned medium, called the enzyme extract, was clarified by centrifugation and filtration. The enzyme reaction was completed in 30 min and was positively correlated with the amount of extract added, until reaching color saturation. Bacillus pumilus str. F166 was the most active isolate and Bacillus cereus str. F65 was about half as active as the positive control. Bacillus cereus str. F27 performed well too. We also have demonstrated strong keratinolytic activity against raw wool α-keratin. Juncos are long-distance migrant birds that carry bacteria on their feathers, both beneficial and harmful to plants. The abundant keratinolytic bacteria on feathers are capable of structurally damaging the feathers, potentially reducing bird fitness and reproductive success. Keratinases have uses in tanning, silage, and remediation. Keratinases may be useful in the treatment of onychomycoses and actinic keratoses in medical and veterinary settings.

Genome Sequence and Analysis of the Flavinogenic Yeast Candida membranifaciens IST 626.

The ascomycetous yeast Candida membranifaciens has been isolated from diverse habitats, including humans, insects, and environmental sources, exhibiting a remarkable ability to use different carbon sources that include pentoses, melibiose, and inulin. In this study, we isolated four C. membranifaciens strains from soil and investigated their potential to overproduce riboflavin. C. membranifaciens IST 626 was found to produce the highest concentrations of riboflavin. The volumetric production of this vitamin was higher when C. membranifaciens IST 626 cells were cultured in a commercial medium without iron and when xylose was the available carbon source compared to the same basal medium with glucose. Supplementation of the growth medium with 2 g/L glycine favored the metabolization of xylose, leading to biomass increase and consequent enhancement of riboflavin volumetric production that reached 120 mg/L after 216 h of cultivation. To gain new insights into the molecular basis of riboflavin production and carbon source utilization in this species, the first annotated genome sequence of C. membranifaciens is reported in this article, as well as the result of a comparative genomic analysis with other relevant yeast species. A total of 5619 genes were predicted to be present in C. membranifaciens IST 626 genome sequence (11.5 Mbp). Among them are genes involved in riboflavin biosynthesis, iron homeostasis, and sugar uptake and metabolism. This work put forward C. membranifaciens IST 626 as a riboflavin overproducer and provides valuable molecular data for future development of superior producing strains capable of using the wide range of carbon sources, which is a characteristic trait of the species.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

Ascorbic acid and all-trans retinoic acid promote proliferation of chicken blastoderm cells (cBCs) by mediating DNA demethylation.

Chicken blastoderm cells (cBCs) obtained from stage X (EG&K) embryos are easily available materials for the study of cell development. However, cBCs are not widely used because they are hard to maintain in long-term culture in vitro. To solve this problem, ascorbic acid (AA; also known as vitamin C (VC)) and all-trans retinoic acid (ATRA) were added into basic culture medium to promote cell growth. Results suggested that cultured cBCs possessed strongly proliferative activity and maintained their pluripotency on the support of chicken embryonic fibroblast (CEF) feeder. Moreover, when VC or/and ATRA was added, the number and area of cBC colonies increased significantly compared with the control group. The expression of pluripotency genes (Sox2 and Nanog) and cell cycle-regulated genes (CCND1 and CDK6) was upregulated obviously. Furthermore, results showed that 5hmC levels in VC and RA groups increased significantly by DNA dot blot and immunofluorescence staining. These results provide strong evidence that VC and ATRA induced DNA demethylation and enhanced 5hmC level. The level of H3K27me3 was raised, while the level of H3K9me2 was reduced by addition of VC and ATRA. Finally, the expression of Tet1 and Dnmt3b was upregulated remarkably. Therefore, these results indicated that VC and ATRA enhanced DNA demethylation and then promoted cBC survival and proliferation in vitro.